PROJECT SUMMARY/ABSTRACT The development of effective therapies for cancer requires a deep molecular understanding of tumor heterogeneity by advanced omics technologies with spatially resolved measurements. Unfortunately, there are substantial gaps in existing capabilities in terms of sensitivity. For example, a minimum of many thousands of cells is required for in-depth profiling of proteins in a biological sample. We have recently developed a breakthrough technology, termed nanoPOTS (Nanodroplet Preparation in One pot for Trace Samples) which, when coupled to ultrasensitive liquid chromatography-mass spectrometry (LC-MS), enables effective analysis of as few as 10 mammalian cells with a coverage of >3000 identified proteins. We hypothesize that the nanoPOTS platform will be an enabling technology to characterize the molecular underpinnings of tumor heterogeneity by creating 3D proteome maps of human tumors. The overall objective of our study is to extend and validate this analysis platform to enable single-cell resolution measurements at high throughput (>100 samples per day) and apply the platform to create 3D proteome maps of human tumors. Studies in Aim 1 will focus on optimizing and validating the ultrasensitive nanoPOTS proteomic workflow to enable robust proteome profiling of ?3,000 protein groups from single human cells obtained by both flow cytometry and laser capture microdissection (LCM). Aim 2 will evaluate and compare two complementary technologies for increasing measurement throughput to ~100 cells/day with minimal impact on proteome coverage. We will determine whether multiplexing through application of isobaric labels for pooled analysis, or rapid LC coupled with ultrahigh resolution ion mobility spectrometry-MS provides greatest coverage, quantitation and reproducibility for single cell proteomics at the desired throughput. In Aim 3, we will apply the optimized platform to create in-depth proteome maps for human pancreatic ductal adenocarcinomas (PDAs) at single cell resolution. Following initial targeted studies of specific cells of interest within sectioned tissues, we will use a combination of cryosectioning, LCM and the optimized nanoPOTS platform to analyze single cells within the tumor microenvironment. 3D reconstruction of these in-depth, spatially resolved proteomic analyses will provide the first global proteomic tumor maps at single-cell resolution. This project will not only establish an innovative measurement capability that will broadly advance cancer research, but will also provide unique molecular insights into cellular heterogeneity relevant to PDA pathology. The resulting platform will be disseminated through a combination of publication and commercialization.